Experiment 3: Microbial Techniques

Experiment 3: Microbial Techniques Objectives: By the end of this lab, you will be able to: 1. Understand and practice aseptic techniques in handling microorganisms. 2. Learn simple media preparation procedures 3. Streak plates and spread plates to obtain single colonies. 4. Understand how serial dilutions are made and how cell density can be determined through standard plate count. 5. Learn to use micropipettes and other types of pipets with pipet pumps. 6. Understand the concept of replica plating. Note: At the end of the lab, your agar plates will be incubated at 30 C for approximately 48 hours and then placed in a refrigerator. You will complete your lab report after analyzing your plates during next week s lab session. A. Sterile and Disinfection Techniques 1) Sterilizing with moist heat: Moist heat provided by an autoclave or pressure cooker at a pressure of 15 psi (at 121 O C) is an efficient way to sterilize most materials. 2) Sterilizing with dry Heat: Dry materials such as glass and metal may be sterilized in an oven, at 160 O C for two hours. 3) Sterilizing by flaming: The flame from a gas burner effectively sterilizes small glass or metal objects, such as inoculating loops. Flame the metal inoculating loop until it is red hot. In order to prevent killing of the microorganisms, you must remember to always cool the flamed inoculating loop by letting it briefly touch sterilized liquid or solid media. To sterilize metal or glass spreaders, dip the spreader in alcohol and then pass the spreader through the flame to ignite the alcohol. 4) Disinfecting the bench with 70 % alcohol: Remove mold-laden dust, the most common source of contamination, by wiping the bench with 70% alcohol. If your skin is not particularly sensitive to alcohol, wipe your hands with a small amount of alcohol, too. 5) Principles to eliminate or minimize airborne contamination or contamination caused by human skin or secretions: B. Growth Media Keep containers for growing microorganisms, such as Petri-dishes, culturing tubes, covered with lid or cap as much as possible. Don't touch anything that have been sterilized and will be in direct contact with microorganisms, such as the micropipette tips. Wipe down the surface around the experiment with alcohol and minimize air turbulence. Avoid talking, singing, whistling, coughing, or sneezing in the direction of things that should be sterile. Long hair, if not tied back, may be a source of contamination. There are different types of media for growing different types of cells. Bacteria and yeast can be grown on solid media or in liquid media. LB (Luria Broth) media which contains tryptone, yeast extract and NaCl is the most common liquid media for growing bacteria. Solid media have the same chemical ingredients as the liquid media except that they contain an additional solidifying 1

agent, typically agar. The concentration of agar for conventional agar plates is 2% (2 grams of agar per 100 ml of final volume). In making the media, the container should not be filled to more than 2/3 of its capacity. For example, you can prepare no more than 600 milliliters of media in a 1000 milliliter flask. If you fill containers too full they may boil over during the sterilization process. It takes approximately 25 milliliters of media to pour one standard 100 x 15 mm plate (Petri dish). Two experiments in BIOL2281 will use one or more of the following types of media: 1) YED medium (Yeast Extract + Dextrose) will be our standard growth medium for yeast. Red adenine-requiring mutants grow well on this medium and develop the characteristic red pigment. 2) YEAD medium (Yeast Extract + Adenine + Dextrose) contains the same ingredients as YED but has excess adenine added. Red adenine-requiring yeast mutants grow well on this medium. 3) MV medium (Minimal plus Vitamins) is a chemically defined medium, meaning it is made up from the minimum of pure chemical ingredients necessary to support growth of wild type yeast. 4) Selective media contain added nutrients, enzymes or antibiotics (e.g. Ampicillin) that only allow a selected strain to grow. C. Growing Cultures The optimum growth temperature for yeast strains is approximately 30 O C, and for most bacteria it is 37 O C. There are four distinct phases during the growth of a microbial culture in a closed system to which no fresh media are added. 1) LAG Phase: the cells adapt to their new environment. The number of cells does not increase during this time. 2) LOGARITHMIC (LOG) Phase: the number of cells doubles at regular intervals (doubling time). 3) STATIONARY Phase: Growth slows as wastes accumulate and/or nutrients are depleted. During the stationary phase, the rate of cell division is equal to the rate of cell death. 4) DEATH Phase: the number of viable cells decreases. D. Streaking Plates and Spreading Plates 1) Streaking Plates: Many experiments require growing cultures from isolated single colonies. This is easily accomplished by streaking cultures out on an agar plate with a flamed and cooled inoculating loop or with the flat end of a toothpick. The purpose of the technique is to dilute the cells on the surface of the agar in successive, overlapping streaks, until individual cells are separated to form isolated single colonies. Each colony represents daughter cells from a common ancestor cell. 2) Spreading Plates: Spreading plate with a small volume of microbial culture can also yield isolated single colonies. It is typically used to determine cell densities (See section E). The Goal of spreading the cells is to evenly distribute a consistent number of cells so that individual colonies can be counted; not so many that the colonies form a lawn. Sterilized glass or metal spreaders are used to spread out the cells evenly across the plate to ensure good separation. 2

E. Counting Microbial Cell Numbers and Determining Cell Density It is often important and necessary to determine the density or titer of the cell culture, i.e. how many cells are present per ml of culture. The two most widely used methods for counting bacteria or yeast numbers are the standard plate count or the spectrophotometric method. A spectrophotometer is used to measure the amount of light that can be transferred through a liquid. If microbial cells are present, they will scatter the light and reduce the amount that can be transmitted through the solution. Generally, the greater the number of cells in the culture, the more light will be absorbed by the cells (absorbance or optical density at specific wavelength, OD 600 ). The spectrophotometer will report absorbance or optical density of a culture, which indirectly reflects the number of microorganisms. This method is fast and easy to perform, however, it is limited by its sensitivity and accuracy: it is used only if the concentration of cells exceeds 1 million per ml and it only provides an estimate of the population size. Furthermore, this method does not distinguish between living and dead cells. An alternative method for measuring the titer of live cells (viable cells) in a culture is the standard plate count or viable plate count method (See Figure below). Since the bacterial culture or yeast cultures are commonly fairly concentrated in cell number, with titers such as 10 6-10 8 cells/ml, serial 10-fold dilutions are necessary to reduce cell concentrations to a point where isolated single cells can be spread out on a plate in a typical spreading volume of 0.1 ml. A good counting plate should have about between 30-300 colonies to be counted accurately. Fewer than 30 colonies are not statistically reliable and more than 300 colonies may produce colonies too close to each other. Several series of dilutions (e.g. 10-4 to 10-8 ) is normally performed because the exact titer of the microbial culture is usually unknown. To calculate the number of bacteria or yeasts per ml: Number of colonies = # of bacteria/ml (cell density or titer) Dilution X Amount plated For example, the cell titer in the original culture from the following diagram can be determined as: 65 / ( 10-3 X 0.1 ml) = 6.5 X 10 5 cells /ml 3

In performing the serial dilution and subsequent plating of cells onto nutrient agar plates, you will use micropipettes or conventional glass or plastic pipettes. Micropipettes are capable of accurately measuring small volumes such as 0.1 ml, 0.05 ml, or 0.01 ml, etc. In this experiment, you will use the 0.1 ml micropipettes for making serial dilutions and for plating cells on agar plates. These micropipettes use micropipette tips of appropriate sizes to dispense the solution. NEVER USE THE MICROPIPETTORS WITHOUT THE TIPS! (The micropipettes will easily be damaged when liquid gets into its internal parts and they are expensive to repair or replace. Do not hold or leave micropipettes upside down.) The yellow tips are packaged in plastic boxes and they are sterilized by autoclaving. Try to keep the lid of the boxes closed whenever you are not using the tips to prevent airborne contamination. Practice using the micropipette with water until you feel comfortable with it. Follow these steps: Press the small end of the pipettor firmly into the open end of a tip while the tip is still in the box; To draw liquid, slowly depress the plunger until you feel some resistance (the first stop). Immerse the tip in the solution and then slowly release the plunger to draw the solution into the tip; Then place the tip into the liquid you are pipetting into and slowly depress the plunger. This time press it as far as possible to expel all the sample. Remove the tip of the pipettor from the solution, release the plunger, and discard the tip. The conventional pipettes along with pipettte pumps may be used instead of micropipettes for small volumes. These pipettes generally do not have as much accuracy as the micropipettes when very small volume is measured, such as volumes under 1 ml. They are either made of glass or plastics. Glass pipettes can be sterilized by dry heat in an oven or by autoclaving. Plastic, pre-sterilized disposable pipettes are also commercially available. It's never a good practice to use your mouth to draw liquid into a pipette. Not even sterile water! Do not hold or leave pipettes upside down. Practice how to correctly read the volumes in these pipettes and how to correctly draw and expel solution with the pump. 4

F. Replica Plating In some experiments many cultures need to be transferred from one kind of medium to one or several other kinds, to determine the cultures' growth requirements. The method--replica plating -- played a major role in the development of microbial genetics. It will be demonstrated by your instructor in the lab. A cotton velveteen stamp is used to stamp a replica of the pattern of cells growing on one plate to one or several other plates. The apparatus consists of a cylindrical holder for the velveteen that is just the right size to fit inside the bottom of a Petri dish, and a ring to hold the velveteen in place. The velveteen must be sterilized and can be reused by rinsing them out in plain water and then sterilized. During this lab, each group (2 persons) will 1. Prepare sterile YED media (Procedure #1; go to procedure 2 if media prepared by TA) 2. Pour 6 YED plates (three plates by each student) using sterile, disposable Petri dishes. 3. Perform a 10X serial dilution of yeast (wild type strain HA0). 4. Spread 6 YED plates (three plates by each student) with different yeast dilutions. 5. Streak 2 YED plate (one plate by each student) with yeast to culture single colonies using a sterile inoculating loop. Procedures: Disinfecting your bench with alcohol and thoroughly washing your hands. Make sure your work station (for 2 students) has the following items. 2 Permanent marker One Roll of masking tape Two Green Pump for pipettes One white container for used micropipette tips 4 autoclaved test tubes in a blue test tube rack 1 tube of diluted overnight yeast culture (tube A) beaker for ethanol with lid, one striker 4 Sterile 1 ml pipet 1 micropipette ( P200: 20-200µl) 1 sterilized yellow tip box 6 of sterilized petri dishes Two of YED plates (for streaking) One spinner (turntable), one can of sterile 5 ml glass pipet, one Bunsen burner, two metal spreaders, two inoculating loops One master plate (Strain HA0 source plate for streaking, shared by 4 students) 1. Two group will share YED media in a 500 ml flask. The following recipe will prepare 300 milliliters of media. Approximately 25 ml of media is needed for one standard culture dish. 3g yeast extract 6g anhydrous dextrose 6g agar 300 ml Tap water Press Tare after you place an empty weighing boat on the balance for measuring. Add all measured materials to a 500 ml flask. Sterilize the media with an autoclave. 5

2. Each person will pour 3 YED plates. Continue to swirl until the flask is warm (not cool) to the touch. Turn on Bunsen burner. Pour enough media into each culture plate to completely cover the bottom of the dish. Open one dish at a time and cover immediately after pouring. Do not disturb the dishes as you allow the plates to cool and solidify. The YED plates you poured will be used for step 6. Obtain more Petri dishes if you have extra media for more plates (used as a backup if extra practice needed). Immediately after pouring the media, rinse out the flask with hot water. The agar will solidify in the flask if allowed to reach room temperature, thus making cleaning very difficult. 3. Each person will streak 1 YED plate ( prepared by TA ) using a sterile inoculating loop. On the BOTTOM side of your YED plate (do not write in the center area), use a marker to record the following information a) your section and assigned number (on sign-in sheet) b) the strain name (HA0) c) streaking d) date, initials 4. Using this streaking technique to decrease the number of cells on the loop as you streak, so individual colonies will grow near the bottom of the streak after incubation. Flame a wire inoculating loop using Bunsen burner until it is red hot to sterilize the loop. Allow it to cool then transfer cells from a master plate and streak them on a YED plate ( prepared by TA). Reflame the loop before streak cells to a new region (see figure below). Pass your loop through the end of your first streak several times and continue streaking in a tight zigzag pattern to the bottom of the section. Reflame the loop when you finish the last streaking. http://www.towson.edu/~gekpenyo/images/streak.gif 6

5. Each group will perform a 10X serial dilution of yeast culture (wild type strain HA0) using aseptic technique. Your goal is to evenly distribute a consistent number of cells onto a plate so that single colonies can be counted; not to spread so many that the colonies form a lawn. Use small strips of masking tape and a marker to label 4 capped sterile culture tubes with the letters B, C, D& E. (Tube A will be provided with diluted Yeast culture with OD 600 at 0.1.) Turn on Bunsen burner. Pipet 4.5 ml of sterile water into 4 labeled, capped tubes (B, C, D and E) using ONE 5 ml sterile pipet: During each transfer, briefly hold the top of tubes in the flame to kill unwanted microbes. A B C D E 1 ml 4.5 ml 4.5 ml 4.5 ml 4.5 ml Remove 0.5 ml from tube A and put into tube B using a 1 ml disposable sterile pipet. Swirl or use a vortexer to mix right before each transfer. Remove 0.5 ml from B to C, C to D and then D to E. Use new 1 ml disposable sterile pipet for each transfer. Place used glass pipettes in a container near front sink, dispose 1 ml disposable pipettes (without plastic bag) in a jar near biohazard trash can. Question: What is the final volume in each of the tubes after the dilution? A: ; B ; C ; D ; E 6. Labeling your YED plates. On the BOTTOM side of your YED plates (do not write in the center area), use a marker to record the following information a) your section and assigned number (on sign-in sheet) b) the strain name c) spreading with the dilution tube number ( C, D, E) d) date, initials 7. Each person will spread 3 YED plates. You will spread each plate with different titers (from cell suspension C, D or E). Each group should have two plates for dilution tube C, D, and E, respectively. The metal spreader is first sterilized by dipping it into a beaker of alcohol and then holding it in a flame. Cover the ethanol beaker right after the spreader is removed from the beaker. Leave the spreader face up on a TEST TUBE RACK (DO NOT LET THE TRIANGULAR PORTION TOUCH ANYTHING!!!) While the spreader is cooling, use micropipettes and yellow tips to inoculate with 0.1 ml (100 µl) of the yeast culture and dispense it evenly over the agar. Touch the edge of the media with the spreader to make sure the spreader has cooled. Once you are sure that the spreader has cooled, quickly open the cover of the plate and spread the liquid evenly. Hold the lid face down just above the plate instead of putting the lid down on 7

your bench when you spread the liquid. Sterilize the spreader again after you finish spreading. Pour the used ethanol back to the big ethanol container bottle after this procedure. Follow the instructions on board to dispose Yeast culture and to place the tubes. Shut the gas valves after this procedure. 8. Stack your four plates (three spreading and one streaking) and secure them with a single strip of masking tape. Record your section and lab ID number (sign-in sheet) on the tape in large print. A tray will be provided to collect your plates. Turn the stack upside down to prevent condensation from dripping onto the colonies and disrupting the growth pattern. Your plates will be incubated at 30ºC for two days and refrigerated till next lab session. Bring this lab procedure next week again. 9. Your TA will assign 4_ of the 20 points allocated for this lab report based upon the following criteria: 1 point for sterility (no contamination), 1 point for correct labeling, and 2 point for the correct streaking pattern and the growth of single colonies on the streak plate. 10. Lab practical for microbial techniques will be assessed during midterm. A set-up of practical is provided in today s lab session for you to practice. Cleaning Up: a) Turn off the gas valve. b) Dispose the used yellow tips into big biohazard trashcan. Dispose 1 ml disposable pipettes to the tall pipette jar. c) Return Tube A to common bench. d) Recycle the ethanol in glass beaker to the big ethanol bottle. e) Take off the masking tape label from all of your test tubes. Rinse all of the used test tubes with tap water a couple of times, SCRUB with the tube brush, rinse again, place the clean tubes upside down in the metal basket. f) Place equipments and supply to the top of student shelf. Wipe your bench area with 70% ethanol (clear top squirt bottle). 11. The following week: Photograph or illustrate the growth pattern of all plates from your group. Count the number of colonies on your plates (C, D and E). Draw dots on the bottom of each plate using a marker to help you count the colony. For a plate with over 300 colonies, you can count a quadrant of it then multiply the number by four. Record the results of your group in the tables below. 12. The titer of the original tube (A): Read the sample results on next pages. Use the plates with 30-300 colonies to calculate the concentration of viable Yeast cells present in Tube A. You should use the average number if the colony numbers of you and your partner are within 10 % of average number. You may have to use the data from one person due to the large deviation (see group 2). The estimated results 8

may provide the information to determine which plates with more reliable results. Write your calculation on the space under the table. 13. Leave the sheets with your plates on the top of student shelf for TA to check. Student Names C D E Calculate the concentration of viable Yeast cells present in Tube A. 14. Dispose plates in biohazard trash can. Sample results ( for reports and midterm): By following the protocol above, the results were observed after 48 hours incubation and recorded in the tables below. Group 1: C D E 210 in one quadrant 90 35 (840 estimated) 300 in one quadrant (1200estimated) 82 4 What was the titer of the original tube (A)? Answer: 8.6 x10 5 cells/ml. How was it calculated? 1) Choose only data from Plate D. (Why?) 2) Average 90 and 82. 3) The titer of tube D is 86 cells/0.1 ml or 860 cells/ml or 8.6x10 2 cells/ml. 4) Now work backwards and increase 10 fold for C, B and A: Titer of tube C: 8.6x10 3 cells/ml Titer of tube B: 8.6x10 4 cells/ml Titer of tube A: 8.6x10 5 cells/ml Or you can calculate in another way: 86/ ( 10-3 X 0.1 ml) = 8.6x10 5 cells/ml Note: the dilution from A tube to D tube is 1 to 1000 or 10-3, and the amount plated onto plate D is 0.1 ml. 9

Group 2: C D E 200 in one quadrant 90 10 (estimated 800) 300 in one quadrant 8 6 (estimated 1200) What was the titer of the original tube (A)? Answer: 9.0 x10 5 cells/ml. How was it calculated? 1) Choose only data from Plate D with 90. (Why?) 2) The titer of tube D is 90 cells/0.1 ml or 900 cells/ml or 9.0 x10 2 cells/ml. 3) Now work backwards and increase 10 fold for C, B and A: Titer of tube C: 9.0 x10 3 cells/ml Titer of tube B: 9.0 x10 4 cells/ml Titer of tube A: 9.0 x10 5 cells/ml Or you can calculate in another way: 90/ ( 10-3 X 0.1 ml) = 9.0 x10 5 cells/ml Post Lab Report (16 pts total): Include title Page: Your name, date, the title of the experiment. Do not copy any content or table format from your partners. (4pts) Title page (partners names) and methods: Write the methods section concisely in paragraph format and in past tense. The methods should include the preparation of media (ingredients), whole dilution series, streaking and spreading. Describe the methods of sterilization for media and used equipments. Do not list materials or cite lab manual. (7pts) Results: Illustrate the growth patterns on your streak plate or identify your streak plate with labeled photographs. Illustrate or include labeled photographs of all plates that your group spread. Design a table and record the number of colonies on each plate that your group spread. What were the titers of all the tubes in the dilution series? Illustrate and/or show your calculation work. Explain which set of data was used to calculate the titers. Describe the results section also in paragraph format and in past tense. (5pts) Discussion: Analyze and interpret the results. Why did you observe these data and how did these results contribute to your knowledge? What are the problems to affect your results? Assuming that all of the cells you spread actually grew into colonies, how many cells were actually plated on plate D? Was the cell number on plate D (containing 0.1 ml of cell dilution from tube D) ten-fold of those on plate E? What might be the reasons if the results did not demonstrate the relationship of ten-fold dilution (you have to answer this question even your results did demonstrate the relationship of ten-fold dilution)? Review writing guides for scientific paper in the link below: http://abacus.bates.edu/~ganderso/biology/resources/writing/htwsections.html References: Part G : Yeast Laboratory Methods and Materials in The GENE Project ( funded by the National Science Foundation and the Howard Hughes Medical Institute. Genetics Education Network), Department of Physics, Cardwell Hall, Kansas State University Manhattan, KS 66506, http://www.phys.ksu.edu/gene/chapters.html, accessed on July30, 2006 10

Biol2281-E3 Microbial Techniques BIOL 2281 Quiz Samples Describe the purpose of streaking plates. Describe the procedures to sterilize spreaders, and inoculating loops. Describe the purpose of incubating the plates upside down. Calculate the volume of culture required to set up 10X serial dilution. (final volume: 7 ml, how much of the culture should be transferred?) 1 2 Sterile Techniques Moist heat: using an autoclave or pressure cooker at a pressure of 15 psi (121ºC) for most of materials. Dry Heat: glass and metal may be sterilized in an oven, at 160º C for two hours. Flaming: Flame the metal inoculating loop until it is red hot. To sterilize spreaders, dip the spreader in alcohol and then pass it through the flame to ignite the alcohol. Cool the flamed inoculating loop and spreader. Radiation Wiping the bench with 70 % alcohol only disinfecting it 3 4 Principles to minimize airborne contamination Keep containers, Petri-dishes and culturing tubes covered as much as possible. Don't touch anything that have been sterilized such as the micropipette tips. Wipe down the surface around the experiment with alcohol and minimize air turbulence. Avoid talking, singing, whistling, coughing, or sneezing in the direction of things that should be sterile. Tie back long hair. 5 Basic Microbial Techniques Preparing growth media Sterilized by autoclaving or filtration Growing Cultures Transferring Cultures Streaking Spreading Standard plate count (Titering) Pipetting Replica Plating Aseptic techniques Preventing contamination of persons, environment and the specimen 6 Dr. Wenju Lin,Spring 2016 1

Biol2281-E3 Preparing growth media Types of media Liquid or Solid (containing agar) Minimal: Contains basic sugars and salts Rich: Contains basic sugars and salts plus nucleotide precursors amino acids vitamins Minerals Some Basic Media LB broth : primarily used for the growth of bacteria containing tryptone, yeast extract, NaCl, H 2 O formulated by Giuseppe Bertani in 1951 as Lysogeny broth (LB), also known as Luria broth or Luria-Bertani broth. YED : yeast extract plus dextrose is standard for yeast (complete, complex) Selective: Contains nutrients, enzymes, hormones, or drugs (Ampicillin) that allows only a selected strain to grow. An indicator (X-gal) may be added to indicates a phenotypic change. 7 MV : minimal media chemically defined to support growth of yeast; without adenine or other amino acids 8 Growing Cultures Temperature: Incubate bacteria at 37 C,yeast at 30 C Equipment : Incubate liquid culture on roller drum or shaker at 200 rpm *Incubate agar plates (upside down) in incubators *labeling on the bottom plates (not in the center) 9 Bacterial Growth Curve Four distinct phases in a closed system LAG Phase: The number of cells does not increase LOGARITHMIC (LOG) Phase: the number of cells doubles at regular intervals (doubling time). STATIONARY Phase: the rate of cell division is equal to the rate of cell death. DEATH Phase: the number of viable cells decreases. http://www-micro.msb.le.ac.uk/labwork/bact/bact1.htm 10 Bacterial Growth Curve Biological classification (taxonomy) Strain a genetic variant or subtype of a microorganism. Strain, Species, Genus, Family, Order, Class, Phylum, Kingdom Type strain often the first strain isolated or best characterized stored in American Type Culture Collection (ATCC) Bacteriology Collection: 18,000 strains Yeast collection: more than 2,000 genetic strains Colony Each colony represents daughter cells from a common ancestor cell. 11 12 Dr. Wenju Lin,Spring 2016 2

Biol2281-E3 Streaking a plate The purpose of streaking a plate is to dilute the cells to form isolated single colonies. Flame a wire inoculating loop using Bunsen burner to sterilize the loop. Allow it to cool before transfer cells. Reflame the loop before streak cells to a new region. Illustration of streaking for purification. The colors represent the pattern of growth (of the same bacterium) seen in each respective streak-zone. http://www.towson.edu/~gekpenyo/images/streak.gif 13 14 Spreading the cells A metal (or glass) spreader is sterilized by dipping it into a beaker of alcohol l and dthen holding it in a flame. The goal is to dilute the cells to form isolated single colonies. Each colony represents the progeny of a single cell. The spreader should be cooled by touching it to the edge of the agar before using it to spread the cells. Hold the lid facing down Cover the beaker 15 16 Pipetting Types: Micropipettes : used with sterilized tips Glass pipettes : sterilized by autoclaving or baking plastic pipettes : sterilized by manufactures The Goal of spreading the cells is to evenly distribute a consistent number of cells so that individual colonies can be counted; not so many that the colonies form a lawn. 17 Read the instructions of pipetting in the procedures Practice each pipet with water. 18 Dr. Wenju Lin,Spring 2016 3

Biol2281-E3 Determining Cell Density The density (or titer) of the cell culture can be measured indirectly with a spectrophotometer by standard plate count (viable plate count) 19 20 10X Serial Dilution Yeast strain A 0.5 ml of A 0.5 ml of B 0.5 ml of C 0.5 ml of D 4.5 ml dh 2 O B 4.5 ml dh 2 O C 0.1ml 4.5 ml dh 2 O D 0.1 ml 4.5 ml dh 2 O E 0.1ml This set of plates contain a 1 to 10 dilution series. A good counting plate should have about between 30-300 colonies to be counted accurately. 21 Use 0.5 ml from previous tube when Final volume is 5 ml 210 in 1/4 300 in 1/4 90 82 5 4 22 Serial dilution The concentration of each sample decreases by the same quantity in each successive step. Dilution factor: 1/10 :1 part of the previous sample added to 9 parts of diluent. Problem: Final volume is 7 ml in 10 X serial dilution, how much of the previous sample should be added to how much of 23 the diluent? Replica Plating Using a cotton velveteen stamp to imprint a replica of the pattern of cells growing on one plate cells to another (or several others) Illustration from The Gene Project, Kansas State University 24 Dr. Wenju Lin,Spring 2016 4

Biol2281-E3 Sample Results of Replica plating Which colonies are histidine-prototroph and adeninerequiring (auxotroph)? a Rich media Media without histidine Media without adenine b c d e c 25 In the lab Each group of students Perform one set of 10x serial dilution Each student Pour three plates Spreading three plates with different dilutions (from C, D or E) Streak one plate (provided by TA) using inoculating loop Next week: Bring E3 procedures again. Record the results then write your own report based on the group data 26 Results on following week Plates will be incubated at 30ºC for two days and refrigerated till next lab session. Count the number of colonies on your plates. Use markers to draw dots on the bottom of the plates. Use plates with 30-300 colonies to calculate the concentration of viable Yeast cells present in Tube A. Record estimated numbers for plates with colony numbers significantly higher than 300. Count the number of colonies on a fraction of the plate, and then multiplying to estimate the number on the entire plate. 27 Dr. Wenju Lin,Spring 2016 5